Means and methods for influencing the stability of cells

ABSTRACT

The invention provides a method for influencing the stability of an antibody producing cell, comprising directly or indirectly influencing the amount of BCL6 and/or Blimp 1 expression product within said antibody producing cell. Stable antibody producing cells and cell lines are also provided, as well as methods for producing antibodies using such cells and/or cell lines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S.application Ser. No. 12/086,269, filed Jun. 6, 2008, which claimspriority to and the benefit of PCT/NL2005/000848 filed Dec. 9, 2005, theentire contents of all of which are fully incorporated herein byreference.

DESCRIPTION

The invention relates to the field of cell biology.

Ex vivo cell cultures are important tools in current biological andmedical applications. One important application is culturing antibodyproducing cells in order to harvest antibodies, preferably monoclonalantibodies. Monoclonal antibodies (mAbs) represent multiple identicalcopies of a single antibody molecule which copies bind to antigens withthe same affinity and promote the same effector functions. Amongst thebenefits of mAbs is their specificity for the same epitope on anantigen. This specificity confers certain clinical advantages on mAbsover more conventional treatments while offering patients an effective,well tolerated therapy option with generally low side effects. MoreovermAbs are useful for biological and medical research.

The proliferative capacity of most primary cells in culture is limitedby the induction of senescence. This state of irreversible growth-arrestis characterized by expression of a number of senescence-associatedmarkers, such as senescence-associated beta-galactosidase,plasminogen-activator inhibitor 1 (PAI-1), p19^(ARF), p53, p21^(CIP1),and p16^(INK4A). In order to provide a proliferating cell line, cellsare often fused to cancer cells in order to produce hybridoma cells. Theresulting hybridoma cells are capable of dividing indefinitely and growwell in cell culture. Individual hybridomas with a desiredcharacteristic can then be selected for a given purpose.

In order to directly obtain human monoclonal antibodies with a desiredspecificity it would be convenient to isolate a B cell capable ofproducing such antibody and to culture the B cell ex vivo. However,hybridoma technology with human B cells has not been very successfulbecause the resulting hybridomas are unstable. Many attempts for ex vivoculturing of B cells have been undertaken. It is well documented thathuman naive and memory B cells can be cultured for a limited periodfollowing engagement of CD40 in the presence of cytokines, includingIL-2, IL-4 and IL-10 (Banchereau et al., 1991) and it is believed thatthis system mimics the in viva response of B cells towards cognateantigen primed CD40L-expressing helper T cells, in the absence of CD40ligation, IL-10 alone or in combination with IL-2 inducesdifferentiation into antibody-producing cells (Malisan et al., 1996).The mechanisms of regulation of survival and proliferation of mature Bcells cultured under these conditions are only partly known.

Engagement of CD40 on B cells has multiple effects including protectionagainst apoptosis, (partial) inhibition of differentiation and inductionof cytokine responsiveness by B cells. Expression of a large number ofcell cycle inhibitors was decreased by CD40 engagement including Rb-1and Rb-2 (Dadgostar et al., 2002) and it is likely that downregulationof such genes release resting B cells from quiescence. Although CD40triggering leads to a brief proliferative response, cytokines areinstrumental in sustaining cell cycle progression of the triggered Bcells. IL-2 and IL-4 are the most efficient cytokines that promotecontinued cell cycle progression of CD40 or surface Ig-stimulated Bcells. Yet, B cell cultures described in the above mentioned papers areonly stable during a limited period.

Another approach for immortalizing B cells is Epstein-Barr virus (EBV)transformation. However the frequency of B cells that are transformed byEBV is low and therefore attempts to generate EBV transformed B cellsthat produce desired antibodies have met with little success. Recently,Traggiai et al have reported a method for more efficient Epstein-Barrvirus transformation of human B cells that increased the frequency of Bcells that were transformed. With this method B cells obtained from apatient who recovered from severe acute respiratory syndrome coronavirus(SARS-CoV) infection were transformed with EBV and transformed B cellclones that produce monoclonal antibodies specific for SARS and otherviral proteins were isolated (Traggiai et al, 2004).

Yet another approach for immortalizing B cells is described in patentapplication WO 03/052083. This application describes a method ofstabilizing B cells wherein human B cells are transduced withconstitutively active signal transducer of activation and transcription(CA-STAT). A prolonged life span of B cells was observed. Replicating Bcells were however not capable of producing antibody at the same time.Antibodies could be obtained by halting the replication of the cells,thereby bringing about terminal differentiation. The terminallydifferentiated cells produced antibody during a restricted time, afterwhich the differentiated cells died. However, the replicating B cells ofWO 03/052083 lose their capability of developing into antibody producingcells after culturing of 1.5-2 months or longer, rendering these B cellcultures unsuitable for antibody production.

Although various approaches for culturing antibody producing cells havebeen described, there is still a need for means and methods forinfluencing the stability of antibody producing cells. It is an objectof the present invention to provide such means and methods.

Accordingly the invention provides a method for influencing thestability of an antibody producing cell, comprising directly orindirectly influencing the amount of BCL6 and/or Blimp-1 expressionproduct within said antibody producing cell. Preferably the amounts ofboth BCL6 and Blimp-1 expression products within said antibody producingcell are regulated, since both expression products are involved in thestability of an antibody producing cell. The stability of an antibodyproducing cell is defined as the capability of said antibody producingcell to remain in a certain developmental stage (optionally after saidcell has been brought into said stage). Different developmental stagesof a cell involve at least one different characteristic of said cell.For instance, a memory B cell is known to differentiate upon stimulationinto an antibody-secreting plasma cell via a stage which someresearchers call a plasmablast. A memory B cell, a plasmablast and aplasma cell are different developmental stages of a B cell, wherein theB cell has different characteristics. A memory B cell exhibits lowproliferation and antibody secretion. A plasmablast exhibits both higherproliferation and higher antibody secretion levels as compared to amemory B cell, whereas a plasma cell secretes high antibody levels butis not capable of proliferating. These three developmental stages arealso characterised by differences in cell surface markers, as shown inTable 1.

With a method of the invention it has become possible to regulate thereplicative life span of an antibody producing cell. A replicative lifespan of an antibody producing cell is defined herein as the time spanwherein a B cell and its progeny cells are capable of replicating whilemaintaining their capability of producing antibody and/or developinginto a cell that produces antibody. The replicative life span of anantibody producing cell is for instance shortened by forcing anantibody-producing cell to enter another developmental stage. In oneembodiment the replicative life span of an antibody producing cell isshortened by forcing said cell into terminal differentiation. This ischaracterised by increased antibody production and cell cycle arrest.During terminal differentiation cells stop proliferating and eventuallydie. Preferably however the replicative life span of an antibodyproducing cell is prolonged, meaning that said antibody producing cellwill not terminally differentiateor only after a longer period ascompared to the same kind of antibody producing cells that are currentlyused—and continue to proliferate in vitro. According to the invention itis possible to regulate the amount of BCL6 and/or Blimp-1 expressionproduct in an antibody producing cell to such extent that the antibodyproducing cell is brought into, and/or kept in, a predetermineddevelopmental state in which the cells continue to proliferate. With amethod of the invention it has therefore become possible to increase thereplicative life span of an antibody producing cell since it is possibleto maintain a B cell in a certain developmental stage whereinreplication occurs. In current ex vivo B cell cultures the replicativelife span is only a few weeks to two months. After this time thecultured cell lose their capability of replicating, their capability ofproducing antibody and/or their capability of developing into a cellthat produces antibody. With a method according to the current inventionhowever it has become possible to prolong the replicative life span ofantibody producing cells, so that ex vivo cultures are generatedcomprising cells that are capable of replicating and producing antibody(or developing into cells that produce antibody).

An antibody producing cell is defined as a cell which cell is capable ofproducing and/or secreting antibody or a functional part, derivativeand/or analogue thereof, and/or which cell is capable of developing intoa cell which is capable of producing and/or secreting antibody or afunctional part, derivative and/or analogue thereof. Preferably, saidantibody producing cell comprises a B cell and/or a B cell-derivedplasma cell. A B cell is called herein an antibody producing cell, evenwhen the B cell is in a stage wherein antibody production is low or notpresent at all, such as a naive B cell or a memory B cell, beingactivated or not, because such cells are capable of developing intocells that produce antibody, such as a plasmablast and/or plasma cell.

A functional part of an antibody is defined as a part which has at leastone same property as said antibody in kind, not necessarily in amount.Said functional part is preferably capable of binding a same antigen assaid antibody, albeit not necessarily to the same extent. A functionalpart of an antibody preferably comprises a single domain antibody, asingle chain antibody and/or a FAB fragment. A functional derivative oranalogue of an antibody is defined as an antibody which has been alteredsuch that at least one property—preferably an antigen-bindingproperty—of the resulting compound is essentially the same in kind, notnecessarily in amount.

BCL 6 encodes a transcriptional repressor which is required for normal Bcell and T cell development and maturation and which is required for theformation of germinal centers. (Ye, 1997). BCL6 is highly expressed ingerminal center B cells whereas it is hardly expressed in plasma cells.BCL6 inhibits differentiation of activated B cells into plasma cells.The transcriptional repressor B lymphocyte induced maturation protein-1(Blimp-1) is required for development of a B cell into a plasma cell.The human variant of Blimp-1 is named Prdm1. As used herein, anyreference to Blimp-1 includes a reference to Prdm1. Blimp-1 drivesplasma cell differentiation, BCL6 and Blimp-1 repress expression of theother; thus in a natural situation when one reaches an higher expressionlevel than the other, the stage of differentiation is enforced. In thehuman body, differentiation of plasma cells from activated naive ormemory B cells involves downregulation of BCL6 and upregulation ofBlimp-1. In germinal center cells BCL6 expression is high and Blimp-1expression is low. In resting memory cells expression of BCL6 andBlimp-1 are low. Signals that trigger differentiation cause anupregulation of Blimp-1, and this Blimp-1 counteracts the expression ofBCL6. The stage where both BCL6 and Blimp-1 are expressed is short-livedand is called a plasmablast. With progressively increasing Blimp-1levels, BCL6 expression is extinguished, resulting in a plasma cell.

One embodiment provides a method according to the invention wherein BCL6and Blimp-1 are co-expressed in an antibody producing cell (meaning thatboth BCL6 and Blimp-1 are expressed in an antibody producing cell)resulting in an antibody producing cell that is capable of proliferatingwhen an appropriate signal is provided. It has been found thatco-expression of BCL6 and Blimp-1 results in an antibody producing cellwhich is capable of both proliferating and producing antibody. BCL6 andBlimp-1 are preferably co-expressed in a B cell, preferably a human Bcell. Co-expression of BCL6 and Blimp-1 in a B cell results instabilization of said B cell in a plasmablast-like stage. Plasmablasts,like plasma cells, are capable of secreting antibody. However,plasmablasts are still capable of proliferating, whereas plasma cellshave lost their capability of proliferating. Plasma cells are thereforeunsuitable for culturing antibody-producing cell lines. Althoughplasmablasts exert highly favourable proliferating andantibody-producing characteristics, they have not yet been used for longterm antibody production since it has not been possible to stabilizeplasmablasts until the present invention.

With a method of the invention it has amongst other things becomepossible to convert a naive B cell or a memory B cell into aplasmablast-like cell and to stabilize said cell, so that rapiddifferentiation into a plasma cell does not occur. This is contrary tonatural development of plasma cells, wherein expression of Blimp-1 in amemory B cell results in rapid development into a plasma cell, therebyinhibiting BCL6 expression so that the resulting plasma cell hardlyexpresses BCL6. One embodiment of the present invention thus involvesco-expression of both BCL6 and Blimp-1 in a B cell, resulting in a cellthat is capable of both proliferating and producing antibody. Preferablya stable culture of B cells is generated. Stable long term ex vivocultures of antibody producing cells have now become possible.

The amount of BCL6 expression product (preferably a BCL6 protein) in anantibody producing cell is regulated in a variety of ways. In oneembodiment an antibody producing cell is provided with a compoundcapable of directly or indirectly influencing BCL6 expression. Anantibody producing cell is preferably provided with a compound capableof enhancing BCL6 expression, in order to counteract downregulation BCL6during expression of Blimp-1. Such compound preferably comprises aSignal Transducer of Activation and Transcription 5 (STATS) protein or afunctional part, derivative and/or analogue thereof, and/or a nucleicacid sequence coding therefore. STATS is a siatal transducer capable ofenhancing BCL6 expression. There are two known forms of STATS, STAT5aand STAT5b, which are encoded by two different, tandemly linked genes.Administration and/or activation of STATS results in enhanced BCL6levels. Hence, downregulation of BCL6 by Blimp-1 is at least in partcompensated by upregulation expression of BCL6 by STATS or a functionalpart, derivative and/or analogue thereof. Hence, STAT5 or a functionalpart, derivative and/or analogue thereof is capable of directlyinfluencing BCL6 expression. It is also possible to indirectly influenceBCL6 expression. This is for instance done by regulating the amount of acompound which in turn is capable of directly or indirectly activatingSTAT5 and/or regulating STAT5 expression. Hence, in one embodiment theexpression and/or activity of endogenous and/or exogenous STAT5 isincreased. It is for instance possible to indirectly enhance BCL6expression by culturing an antibody producing cell in the presence ofinterleukin (IL) 2 and/or IL 4 which are capable of activating STAT5.

Preferably, an antibody producing cell is provided with a nucleic acidsequence encoding STAT5 or a functional part, derivative and/or analoguethereof, wherein said nucleic acid sequence is constitutively active,meaning that STAT5 is continuously expressed, independent of thepresence of (endogenous) regulators. In case that endogenous STAT5expression is low, or absent, an exogenous constitutively active nucleicacid sequence encoding STAT5 or a functional part, derivative and/oranalogue thereof is preferably applied resulting in a concentration ofSTAT5 or a functional part, derivative and/or analogue thereof which issufficient to enhance BCL6 expression. Most preferably, an antibodyproducing cell is provided with a nucleic acid sequence encoding acompound comprising STAT5 or a functional part, derivative and/oranalogue thereof, preferably a fusion protein, whose activity isregulated by an exogenous inducer of repressor, so that the extent ofactivation of BCL6 expression is regulated at will. Another system thatallows for induction of BCL-6 is provided by a Tet-on system in whichaddition of tetracycline and/or derivatives of tetracycline induceactivity of a transactivator that induced BCL6 gene transcriptionfollowed by BCL protein synthesis. In one preferred embodiment, anantibody producing cell is provided with a nucleic acid sequenceencoding an estrogen receptor (ER) and STAT5 as a fusion proteinER-STAT5. This fusion protein is inactive because it forms a complexwith heat shock proteins in the cytosol. This way, STAT5 is unable toreach the nucleus and BCL6 expression is not enhanced. Uponadministration of the exogenous inducer 4 hydroxy-tamoxifen (4HT), thefusion protein ER-STAT5 dissociates from the heat shock proteins, sothat STAT5 is capable of entering the nucleus and activating BCL6expression.

Additionally, or alternatively, BCL6 expression in an antibody producingcell is enhanced by culturing said antibody producing cell in thepresence of a compound capable of directly or indirectly enhancing BCL6expression.

One embodiment therefore provides a method according to the inventioncomprising:

-   providing said antibody producing cell with a compound capable of    directly or indirectly enhancing BCL6 expression; and/or-   culturing said antibody producing cell in the presence of a compound    capable of directly or indirectly enhancing BCL6 expression. Said    compound capable of directly or indirectly enhancing BCL6 expression    preferably comprises STAT5 or a functional part, derivative and/or    analogue thereof. Provided is therefore a method according to the    invention comprising providing said antibody producing cell with    STAT5 or a functional part, derivative and/or analogue thereof, or    with a nucleic acid sequence encoding STAT5 or a functional part,    derivative and/or analogue thereof. In one embodiment said antibody    producing cell is cultured after introduction of a nucleic acid    sequence encoding STAT5 or a functional part, derivative and/or    analogue thereof into said cell. Said nucleic acid sequence is for    instance introduced into said cell by transfection and/or    virus-mediated gene transfer. Many alternative methods for    introducing a nucleic acid sequence into a cell are available in the    art which need no further explanation here.

With a compound capable of directly or indirectly enhancing BCL6expression it is possible to enhance expression of endogenous BCL6. Inone preferred embodiment however an antibody producing cell is providedwith a nucleic acid sequence encoding BCL6 or a functional part,derivative and/or analogue thereof. This way, it is possible to regulatea BCL6 concentration in an antibody producing cell independently fromexpression of endogenous BCL6. Hence, even if expression of endogenousBCL6 is low or absent, for instance caused by Blimp-1, an exogenousnucleic acid sequence encoding BCL6 or a functional part, derivativeand/or analogue thereof is still capable of producing a concentration ofBCL6 which is sufficient for influencing the stability of an antibodyproducing cell. Also provided is therefore a method according to theinvention comprising providing said antibody producing cell with anucleic acid sequence encoding BCL6 or a functional part, derivativeand/or analogue thereof. Preferably, said antibody producing cell isprovided with a constitutively active nucleic acid sequence encodingBCL6 or a functional part, derivative and/or analogue thereof, so thatBCL6 expression is maintained even when endogenous BCL6 expression ofsaid cell is inhibited by an endogenous repressor such as Blimp-1. Mostpreferably, expression of said nucleic acid sequence encoding BCL6 or afunctional part, derivative and/or analogue thereof is regulated by anexogenous inducer of repressor, so that the extent of BCL6 expression isregulated at will. For instance, an inducible promoter system is usedsuch as a Teton or Tet-off system.

The amount of Blimp-1 expression in an antibody producing cell is alsoregulated in a variety of ways. In one embodiment an antibody producingcell is provided with a compound capable of directly or indirectlyinfluencing Blimp-1 expression. Additionally, or alternatively, anantibody producing cell is cultured in the presence of a compoundcapable of directly or indirectly influencing Blimp-1 expression.Further provided is therefore a method according to the inventioncomprising providing said antibody producing cell with a compoundcapable of directly or indirectly influencing Blimp-1 expression.Further provided is a method according to the invention comprisingculturing said antibody producing cell in the presence of a compoundcapable of directly or indirectly influencing Blimp-1 expression.Preferably, a compound is used that is capable of enhancing Blimp-1expression in order to counteract downregulation of Blimp-1 duringexpression of BCL6. Said compound most preferably comprises IL21.

In one preferred embodiment said compound capable of directly orindirectly influencing Blimp-1 expression comprises a Signal Transducerof Activation and Transcription 3 (STAT3) protein or a functional part,derivative and/or analogue thereof, and/or a nucleic acid sequencecoding therefore. STAT3 is a signal transducer which is involved in Bcell development and differentiation. STAT3 is capable of upregulatingBlimp-1 expression. Further provided is therefore a method according tothe invention wherein said compound capable of directly or indirectlyinfluencing Blimp-1 expression comprises STAT3 or a functional part,derivative and/or analogue thereof, or a nucleic acid sequence encodingSTAT3 or a functional part, derivative and/or analogue thereof. Mostpreferably, expression of said nucleic, acid sequence encoding STAT3 ora functional part, derivative and/or analogue thereof is regulated by anexogenous inducer of repressor, so that the extent of STAT3 expressionis regulated at will. For instance, an inducible promoter system is usedsuch as for instance a Tet-on or Tet-off system. In one embodiment afusion product comprising of STAT3, a derivative or analogue, and ER isintroduced in said cell allowing regulation of STAT3 expression byhydroxytamoxifen.

Since STAT3 is capable of influencing Blimp-1 expression, it is alsopossible to indirectly regulate Blimp-1 expression by administering acompound capable of directly or indirectly regulating the activityand/or expression of STAT3. In one embodiment an antibody producing cellis provided with a compound that is capable of enhancing the activity ofSTAT3, so that Blimp-1 expression is indirectly enhanced as well.Further provided is therefore a method according to the invention,wherein an antibody producing cell is provided with a compound capableof directly or indirectly enhancing activity of STAT3.

Hence, in one embodiment an antibody producing cell is provided with acompound capable of directly or indirectly activating STAT3, in order toenhance Blimp-1 expression.

STAT3 is activated in a variety of ways. Preferably, STAT3 is activatedby providing an antibody producing cell with a cytokine. Cytokines,being naturally involved in B cell differentiation, are very effectivein regulating STAT proteins. Very effective activators of STAT3 areIL-21 and IL-6, but also IL-2, IL-7, IL-10, IL-15 and IL-27 are known toactivate STAT3. Moreover, Toll-like receptors (TLRs) which are involvedin innate immunity are also capable of activating STAT3. One embodimenttherefore provides a method of the invention, wherein said compoundcapable of directly or indirectly influencing Blimp-1 expressioncomprises IL-21, IL-2, IL-6, IL-7, IL-10, IL-15 and/or IL-27. Mostpreferably IL-21 is used, since IL-21 is particularly suitable forinfluencing the stability of an antibody producing cell IL-21 is capableof upregulating Blimp-1 expression even when Blimp-1 expression iscounteracted by BCL6.

Additionally, or alternatively, a mutated Janus kinase (JAK) is used inorder to activate STAT3. Naturally, a JAK is capable of phosphorylatingSTAT3 after it has itself been activated by at least one cytokine. Amutated Janus kinase capable of activating STAT3, independent of thepresence of cytokines, is particularly suitable in a method according tothe present invention.

As already explained before, a compound capable of enhancing Blimp-1expression in one embodiment comprises a nucleic acid sequence encodingSTAT3 or a functional part, derivative and/or analogue thereof. Thepresence of an exogenous nucleic acid sequence encoding STAT3 or afunctional part, derivative and/or analogue thereof allows for acontinuous presence of STAT3 or a functional part, derivative and/oranalogue thereof even when expression of endogenous STAT3 is very low orabsent.

It is also possible to decrease expression and/or activity of STAT5 inorder to upregulate Blimp-1. If the amount and/or activity of STAT5 isdecreased, activation of BCL6 expression is decreased as well resultingin a decreased amount of BCL6 expression product. Since BCL6 and Blimp-1counteract each other's expression, a decreased amount of BCL6expression product results in an increased amount of Blimp-1 expressionproduct. Compounds capable of downregulating the activity of STAT5 arethus capable of indirectly upregulating Blimp-1. Such compounds forinstance comprise members of the suppressor of cytokine signaling (SOCS)proteins. In one embodiment the amount of Blimp-1 expression product inan antibody producing cell is therefore upregulated by providing saidcell with a SOCS protein, and/or by activating a SOCS protein withinsaid cell.

By at least a functional part of a STAT5 protein, a STAT3 protein and/orBCL6 is meant a proteinaceous molecule that has the same capability—inkind, not necessarily in amount—of influencing the stability of anantibody producing cell as compared to a STAT5 protein, a STAT3 proteinand/or BC16, respectively. A functional part of a STAT5 protein or STAT3protein is for instance devoid of amino acids that are not, or only verylittle, involved in said capability. A derivative of a STAT5 protein, aSTAT3 protein and/or BCL6 is defined as a protein which has been alteredsuch that the capability of said protein of influencing the stability ofan antibody producing cell is essentially the same in kind, notnecessarily in amount. A derivative is provided in many ways, forinstance through conservative amino acid substitution wherein one aminoacid is substituted by another amino acid with generally similarproperties (size, hydrophobicity, etc), such that the overallfunctioning is likely not to be seriously afffected. A derivative forinstance comprises a fusion protein, such as a STATS-ER fusion proteinwhose activity depends on the presence of 4 hydroxy-tamoxifen (4HT). Ananalogue of a STAT5 protein, a STAT3 protein and/or BCL6 is defined as amolecule having the same capability of influencing the stability of anantibody producing cell in kind, not necessarily in amount. Saidanalogue is not necessarily derived from said STAT5 protein, STAT3protein and/or BCL6.

A method according to the invention is particularly suitable forproducing an antibody producing cell culture comprising antibodyproducing cells that are capable of proliferating and secretingantibody. In one embodiment, a memory B cell is used in order to producean ex vivo B cell culture. Alternatively, or additionally, a naive Bcell is used. Said memory B cell and/or naive B cell is preferably humanso that human antibodies are produced. Preferably a memory B cell isused with a desired specificity. This means that a memory B cell is usedwhich is capable of developing into an antibody secreting cell, whichantibodies have a desired specificity against an antigen of interest. Inone embodiment B cells are isolated from a peripheral blood sample, acord blood sample and/or a tonsil sample, using methods known in theart. Memory B cells are for instance isolated by selection for the Bcell marker CD19 and (subsequent) selection for cell surface IgG and/orCD27. In a germinal center B cell, BCL6 expression is high whereasBlimp-1 expression is low. Natural development into an antibodysecreting cell involves upregulation of Blimp-1 expression. SinceBlimp-1 represses BCL6 expression, upregulation of Blimp-1 results indownregulation of BCL6 in a natural situation. In a preferred embodimentof the present invention however, Blimp-1 expression is upregulatedwhile BCL6 expression is at least in part maintained. This results in anantibody producing cell wherein BCL6 and Blimp-1 are co-expressed. Saidantibody producing cell is capable of proliferating and secretingantibody and is therefore suitable for use in an ex vivo B cell culture.An antibody producing cell according to the present invention providesthe advantage that it is stable and does not undergo terminaldifferentiation. Said antibody producing cell according to the inventionis stable for at least one week, preferably for at least one month, morepreferably for at least three months, most preferably for at least sixmonths. A B cell according to the invention is preferably cultured inthe presence of CD40L since replication of most B cells is favoured byCD40L.

In one embodiment BCL6 expression is maintained at essentially the samelevel, or at a higher level, as compared to a germinal center B cellsince a significant BCL6 expression, together with Blimp-1 expression,results in an antibody producing cell with preferred proliferation andantibody production properties and/or stability.

One embodiment therefore provides a method for producing an antibodyproducing cell which is stable for at least one week, preferably for atleast one month, more preferably for at least three months, morepreferably for at least six months, the method comprising:

-   providing a memory B cell or a naive B cell;-   increasing an expression level of Blimp-1 in said cell; and-   increasing and/or maintaining a BCL6 expression level in said cell.    Most preferably a memory B cell is used. Said expression level is    preferably brought to, and/or maintained at, essentially the same    level, or at a higher level, as compared to a plasmablast.

Blimp-1 expression and BCL6 expression are influenced in various ways,as already described herein before. For instance, Blimp-1 expression isenhanced in a memory B cell and/or a naive B cell by providing said Bcell with a compound capable of directly or indirectly enhancing Blimp-1expression, such as a nucleic acid sequence encoding Blimp-1 or afunctional part, derivative and/or analogue thereof, and/or a nucleicacid sequence encoding STAT3 or a functional part, derivative and/oranalogue thereof. Preferably, expression of said nucleic acid isregulated by an exogenous inducer of repressor, so that the extent ofBlimp-1 expression is regulated at will.

Alternatively, or additionally, a memory B cell and/or a naive B cell iscultured in the presence of a compound capable of directly or indirectlyenhancing Blimp-1 expression, such as for instance IL-21, IL-2, IL-6,IL-7, IL-10, IL-15, IL-27 or a mutated Janus kinase. Preferably, IL-21is used because this cytokine is particularly suitable for enhancingBlimp-1 expression and stabilizing an antibody producing cell with amethod according to the present invention. In one embodiment a B cell isprovided with a SOCS protein or a functional part, derivative and/oranalogue thereof, or a nucleic acid coding therefore, since a SOCSprotein or a functional part, derivative and/or analogue thereof iscapable of indirectly enhancing Blimp-1 expression. Expression ofBlimp-1 results in downregulation of endogenous BCL6. Therefore, saidmemory B cell is preferably also provided with a compound capable ofmaintaining BCL6 expression, resulting in co-expression of both BCL6 andBlimp-1. Said compound is preferably capable of inducing and/ormaintaining BCL6 expression at essentially the same level or at a higherlevel as compared to a plasmablast. A preferred example of such compoundis a nucleic acid sequence encoding BCL6 or a functional part,derivative and/or analogue thereof.

It is possible to directly provide a B cell with a compound capable ofdirectly or indirectly enhancing BCL6 expression, for instance bytransduction with a nucleic acid sequence. In one embodiment BCL6expression in a B cell is maintained and/or enhanced by culturing amemory B cell in the presence of a compound which is capable of directlyor indirectly enhancing BCL6 expression and/or which is capable ofmaintaining BCL6 expression at essentially the same level, or at ahigher level, as compared to a germinal center B cell.

In a preferred embodiment Blimp-1 expression is upregulated in a B cell,preferably by culturing said B cell in the presence of a compoundcapable of activating STAT3 and/or Blimp-1. Said compound preferablycomprises IL-21. Said B cell preferably comprises a memory B cell. Afterthis, BCL6 expression is preferably enhanced. It has been demonstratedthat Blimp-1 upregulation in a first stage followed by BCL6 upregulationresults in particularly stable B cells capable of replicating andproducing antibody. In one embodiment of the invention Blimp-1expression is still upregulated while BCL6 expression is enhanced.Alternatively however, Blimp-1 expression is not upregulated while BCL6expression is enhanced. This way, the replication capacity of a B cellis particularly enhanced. Hence, an antibody producing capacity of a Bcell is preferably enhanced firstly, by upregulating expression and/oractivity of Blimp-1. Subsequently, a replication capacity of said B cellis preferably enhanced, by upregulating expression and/or activity ofBCL6. The B cell is preferably cultured in the absence of a compoundcapable of enhancing Blimp-1 expression and/or activity, untilreplication is significantly increased. Subsequently, said B cell ispreferably cultured again in the presence of an enhancer of Blimp-1expression and/or activity, so that antibody production is maintained.As is shown in the examples, it is possible to regulate Blimp-1 and BCL6in various ways, resulting hi co-expression of both Blimp-1 and BCL6 ina B cell which B cell is capable of replicating and producing antibody.

In one preferred embodiment Blimp-1 expression is upregulated in a Bcell, preferably a memory B cell, by culturing said B cell in thepresence of a compound capable of activating STAT3. Said compoundpreferably comprises IL-21. According to one embodiment, said B cell issubsequently provided with a nucleic acid sequence encoding BCL6 or afunctional part, derivative and/or analogue thereof. Said B cells arepreferably cultured for a few days in the absence of said compoundcapable of activating STAT3 in order to enhance replication.Subsequently, said cells are preferably again cultured with—and/orprovided with—a compound capable of activating STAT3.

In the Examples a particularly preferred embodiment is shown, wherein Bcells are firstly cultured in the presence of IL-21. Subsequently the Bcells are provided with a nucleic acid sequence encoding BCL6. The Bcells are cultured in the absence of IL-21 for a few days in order toenhance replication, after which IL21 is administered again to theculture in order to enhance antibody production, Stable B cells areobtained wherein BCL6 and Blimp-1 are co-expressed, which B cells arecapable of replicating and producing antibody in an ex vivo cultureduring at least 6 months. A B cell culture according to the invention ispreferred since the B cells are capable of replicating and producingantibody in an ex vivo culture during a longer period of time ascompared to current B cell cultures.

In another preferred embodiment a nucleic acid sequence encoding STATSor a functional part, derivative and/or analogue thereof is used inorder to enhance BCL6 expression.

Prior art attempts to use STAT5 in order to obtain a stable B cellculture capable of producing antibodies, such as described in WO03052083, failed because the B cells lose their capability of developinginto antibody producing cells within 2 months. The present inventionhowever provides the insight that STATS is indeed suitable for producinga stable B cell culture if Blimp-1 expression is upregulated in the Bcells as well. Preferably, Blimp-1 expression in a cell is enhanced,after which BCL6 expression is enhanced by STAT5 or a functional part,derivative and/or analogue thereof.

In a preferred embodiment Blimp-1 expression is upregulated in a Bpreferably by culturing said B cell in the presence of a compoundcapable of activating STAT3. Said compound preferably comprises IL-21,Said B cell preferably comprises a memory B cell. Subsequently, said Bcell is provided with a nucleic acid sequence encoding STAT5 or afunctional part, derivative and/or analogue thereof. Said nucleic acidsequence preferably encodes a compound comprising STAT5 or a functionalpart, derivative and/or analogue thereof, whose activity depends on thepresence or absence of an exogenous regulator. Most preferably said Bcell is provided with a nucleic acid sequence encoding a STAT5-ER fusionprotein whose activity depends on the presence of 4 hydroxy-tamoxifen(4HT). In the resulting B cells, which are capable of both replicatingand producing antibody, Blimp-1 and BCL6 are co-expressed. Once aculture comprising B cells according to the invention has been produced,it is possible to further regulate the replication and antibodyproduction capacity of the B cells by regulating BCL6 and Blimp-1expression. The amount of BCL6 and Blimp-1 expression product isregulated at will during further culturing. For instance, when antibodyproduction of the cells diminishes, the activity of STAT5 is preferablydiminished (preferably by depriving the cell culture of 4hydroxy-tamoxifen) while said B cells are cultured in the presence of acompound capable of activating (expression of) STAT3 and/or Blimp-1.Preferably, the cells are cultured for a while (typically about a fewdays) in the presence of IL-21 and in the absence of 4hydroxy-tamoxifen. When antibody production has been enhanced, culturingis preferably continued in the presence of hydroxy-tamoxifen and in theabsence of said compound capable of activating STAT3 in order to enhancereplication and to make sure that Blimp-1 expression does not completelyabolish BCL6 expression.

In the Examples a particularly preferred embodiment is shown wherein Bcells are firstly cultured in the presence of IL-21 during a few days.Blimp-1 expression is induced and the B cells differentiate intoantibody producing cells. Subsequently the B cells are provided with anucleic acid sequence encoding STAT5-ER. The B cells are cultured in thepresence of 11-21 for about 1-50 days, preferably about 1-30 days, morepreferably about 1.5-21 days, where after the B cells are cultured inthe absence of IL-21 and in the presence of 4-HT in order to activateSTAT5. During this period, BCL6 expression is enhanced in order tomaintain an equilibrium wherein BCL6 and Blimp-1 are co-expressed.Subsequently, IL-21 is administered again to the culture and 4HT iswithdrawn in order to increase Blimp-1 expression. Said equilibriumwherein BCL6 and Blimp-1 are co-expressed is maintained by varying theamount of IL-21 and 4-HT in the culture medium so that both BCL6expression and Blimp-1 expression are maintained. Stable B cells areobtained which are capable of replicating and producing antibody in anex vivo culture during at least 6 months.

Hence, a method of the invention allows for subtle regulation of thereplication capacity and antibody producing capacity of B cells culturedex vivo. When upregulation of antibody production is desired, Blimp-1expression is favored over BCL6 expression. When upregulation ofreplication is desired, BCL6 expression is favored over Blimp-1expression. A method of the invention allows maintenance of anequilibrium wherein BCL6 and Blimp-1 are co-expressed, resulting inantibody producing cells which are capable of replicating and producingantibody ex vivo.

The invention therefore provides a method for producing an antibodyproducing cell which is stable for at least one week, preferably atleast one month, more preferably at least three months, more preferablyat least six months, the method comprising:

-   providing a B cell with a compound capable of directly or indirectly    enhancing Blimp-1 expression and/or culturing a B cell in the    presence of a compound capable of directly or indirectly enhancing    Blimp-1 expression; and-   providing said B cell with a compound capable of directly or    indirectly enhancing BCL6 expression or with a compound capable of    maintaining BCL6 expression at essentially at a higher level, as    compared to a germinal center B cell.

Alternatively, or additionally, said B cell is cultured in the presenceof a compound capable of directly or indirectly enhancing Blimp-1expression, in the presence of a compound capable of directly orindirectly enhancing BCL6 expression, and/or in the presence of acompound capable of maintaining BCL6 expression at essentially the samelevel, or at a higher level, as compared to a natural memory B cell.

Said compound which is capable of directly or indirectly enhancing BCL6expression and/or maintaining BCL6 expression at a higher level, ascompared to a natural memory B cell, preferably comprises:

-   a nucleic acid sequence encoding BCL6 or a functional part,    derivative and/or analogue thereof, and/or-   a nucleic acid sequence encoding STAT5 or a functional part,    derivative and/or analogue thereof, and/or-   a compound capable of directly or indirectly activating STATS,    and/or-   a compound capable of directly or indirectly enhancing expression of    STAT5.

A B cell is preferably firstly provided with a compound capable ofdirectly or indirectly enhancing Blimp-1 expression, and/or cultured inthe presence of a compound capable of directly or indirectly enhancingBlimp-1 expression, before BCL6 expression and/or BCL6 activity of saidB cell is increased.

As already explained herein before, said compound capable of directly orindirectly enhancing Blimp-1 expression preferably comprises IL-21,IL-2, IL-6, IL-7, IL-10, IL-15, IL-27, a SOCS protein, a mutated Januskinase and/or a nucleic acid sequence encoding STATS or a functionalpart, derivative and/or analogue thereof. Most preferably, IL-21 isused. If use is made of a nucleic, acid sequence encoding BCL6, STAT5and/or STAT3, or a functional part, derivative and/or analogue of BCL6,STAT5 and/or STAT3, expression of said nucleic acid sequence ispreferably regulated by an activator and/or repressor that is inducibleby an exogenous compound. This way, expression of said nucleic acidsequence is regulated by determining the amount of exogenous compoundthat is administered.

One embodiment provides a method according to the invention furthercomprising selecting and/or isolating an antibody or a functional part,derivative and/or analogue of interest. In one embodiment IgM producingcells and IgG producing cells are selected and/or isolated. Preferablyan IgG producing cell is selected and/or isolated.

Antibody producing cells generated with a method according to theinvention are suitable for producing antibodies against an antigen ofinterest. In one preferred embodiment however, the genes encoding the Igheavy and/or light chains are isolated from said cell and expressed in asecond cell, such as for instance cells of a Chinese hamster ovary (CHO)cell line. Said second cell, also called herein a producer cell, ispreferably adapted to commercial antibody production. Proliferation ofsaid producer cell results in a producer cell line capable of producingantibody. Preferably, said producer cell line is suitable for producingcompounds for use in humans. Hence, said producer cell line ispreferably free of pathogenic agents such as pathogenic micro-organisms.

A method according to the invention is preferably used for generating anantibody producing cell that is stable for at least one week, preferablyat least one month, more preferably at least three months, morepreferably at least six months so that commercial antibody productionhas become possible. One preferred embodiment provides a methodaccording to the invention, wherein an antibody producing cell isproduced that is capable of producing antibodies against an antigen ofinterest. Most preferably a stable cell line capable of producingmonoclonal antibodies is produced. This is preferably performed by usingmemory B cells that have for instance been isolated from a sample byselection for CD19 (B cell marker) and cell surface IgG and/or CD27 (tomark memory cells). Furthermore, an antibody producing cell capable ofspecifically binding an antigen of interest is for instance selected ina binding assay using said antigen of interest. Subsequently, accordingto this preferred embodiment Blimp-1 and BCL6 are co-expressed in saidantibody producing cell, resulting in a culture of cells capable ofspecifically binding said antigen of interest. If only one memory cellis used, a cell line according to the invention producing monoclonalantibodies is obtained. It is also possible to generate a monoclonalantibody producing cell line starting with various B cells capable ofproducing antibody against different antigens. After a stable B cellculture has been produced with a method according to the invention, a Bcell capable of producing antibodies against a specific antigen ofinterest is isolated and at least a functional part of a gene encodingthe Ig heavy chain and/or light chain from said B cell is expressed in asecond cell line. Preferably at least a functional part of the geneencoding the Ig heavy chain and at least a functional part of the geneencoding the Ig light chain from said B cell are expressed in a secondcell line.

In one embodiment an antibody producing cell, preferably but notnecessarily a memory B cell, that has been obtained from an individualwhich had been previously exposed to an antigen of interest, is used ina method according to the invention. This way, it has become possible toproduce human antibodies of interest ex vivo.

The invention furthermore provides an antibody producing cell which isstable for at least one week, preferably for at least one month, morepreferably for at least three months, more preferably for at least sixmonths, meaning that an antibody producing cell according to the presentinvention is capable of both replicating and producing antibody, orcapable of replicating and developing into a cell that producesantibody, during said time periods. Antibody producing cells accordingto the invention comprise, amongst other things, cells producing IgM orcells each producing other immunoglobulin isotypes like IgG, IgA, IgE.An antibody producing cell according to the invention is particularlysuitable for use in an antibody producing cell line. Antibody producingcells according to the invention are preferably cultured ex vivo andantibody is preferably collected for further use. Antibodies orfunctional parts, derivatives and/or analogues thereof produced with amethod according to the invention are useful for a wide variety ofapplications, such as for instance therapeutic, prophylactic anddiagnostic applications, as well as research purposes and ex vivoexperiments. For instance, a screening assay is performed whereinantibodies or functional parts, derivatives and/or analogues accordingto the invention are incubated with a sample in order to determinewhether an antigen of interest is present.

An antibody producing cell according to the invention preferablycomprises a human cell, producing human antibody, because humanantibodies are more suitable for therapeutic and/or prophylacticapplications in human individuals.

An antibody producing cell according to the invention preferablycomprises an exogenous compound which is capable of directly orindirectly influencing BCL6 expression and/or an exogenous compoundwhich is capable of directly or indirectly influencing Blimp-1expression. An antibody producing cell according to the inventionpreferably comprises an exogenous compound which is capable of directlyor indirectly enhancing BUG expression and/or an exogenous compoundwhich is capable of directly or indirectly enhancing Blimp-1 expression,because co-expression of BCL6 and Blimp-1 results in a preferredantibody producing cell according to the invention which is capable ofproliferating and producing antibody.

As explained herein before, BCL6 expression is enhanced in a variety ofways. BCL6 expression is preferably upregulated using a nucleic acidsequence encoding BCL6 and/or STAT5, or a functional part, derivativeand/or analogue of BCL6 and/or STAT5. Further provided is therefore anantibody producing cell according to the invention, comprising anexogenous nucleic acid sequence encoding BCL6 or a functional part,derivative and/or analogue thereof, and/or an exogenous nucleic acidsequence encoding STAT5 or a functional part, derivative and/or analoguethereof.

Moreover, Blimp-1 expression is enhanced in a variety of ways.Preferably a nucleic acid sequence encoding STAT3 or a functional part,derivative and/or analogue thereof is used. The invention thereforefurther provides an antibody producing cell according to the inventioncomprising an exogenous nucleic acid sequence encoding STAT3 or afunctional part, derivative and/or analogue thereof.

In one embodiment said nucleic acid sequence encoding BCL6, STAT5, STAT3and/or a functional part, derivative and/or analogue of BCL6, STAT5and/or STAT3 is constitutively active, so that BCL6, STAT5, STAT3 and/ora functional part, derivative and/or analogue thereof remains present inan antibody producing cell according to the invention even whenendogenous BCL6, STAT5 and/or STAT3 genes are downregulated byendogenous compounds. Most preferably, expression of said nucleic acidsequence encoding BCL6, STAT5, STAT3 or a functional part, derivativeand/or analogue of BCL6, STAT5 and/or STAT3 is regulated by an activatorand/or repressor that is inducible by an exogenous compound, so that theamount of BCL6, STAT5, STAT3 or a functional part, derivative and/oranalogue thereof is regulated at will by regulating the amount ofexogenous compound that is administered. One embodiment thereforeprovides an antibody producing cell according to the invention, whereinexpression of said nucleic acid sequence encoding BCL6, STAT5, STAT3 ora functional part, derivative and/or analogue of BCL6, STAT5 and/orSTAT3, is regulated by an activator and/or repressor that is inducibleby an exogenous compound.

An antibody producing cell according to the present invention with anincreased stability is particularly suitable for the production of an exvivo cell line. The invention therefore further provides a method forproducing an antibody producing cell line comprising:

-   obtaining a stable antibody producing cell with a method according    to the invention, and-   culturing said antibody producing cell ex vivo.

Preferably a B cell line is generated. Most preferably a stable cellline comprising B cells capable of producing antibodies specificallydirected against an antigen of interest is generated. This is preferablydone by obtaining a B cell which is capable of developing into a cellwhich produces antibodies against an antigen of interest. The amount ofBCL6 and/or Blimp-1 expression in said cell is subsequently regulated.Said B cell is preferably obtained from an individual, preferably ahuman individual, who has been exposed to an antigen of interest.

The invention therefore provides a method according to the invention,comprising:

-   obtaining a B cell from an individual who has been exposed to an    antigen of interest,-   producing an antibody producing cell that is stable for at least one    week, preferably at least one month, more preferably at least three    months, more preferably at least six months using said B cell    obtained from said individual in a method according to the    invention, and-   culturing said antibody producing cell ex vivo.

One important application is the production of antibodies that arecapable of specifically binding an antigen of interest. One embodimentof the invention therefore provides a method for producing antibodiescapable of specifically binding an antigen of interest, the methodcomprising:

-   obtaining a B cell capable of differentiating into a B cell which B    cell produces antibodies capable of specifically binding said    antigen of interest,-   producing an antibody producing cell that is stable for at least one    week, preferably at least one month, more preferably at least three    months, more preferably at least six months using said B cell in a    method according to the invention, and-   obtaining antibodies produced by said antibody producing cell.

Said antibody producing cell is preferably further cultured ex vivo inorder to provide a stable cell line capable of producing antibodieswhich are specifically directed towards an antigen of interest. Morepreferably at least a functional part of a gene encoding the Ig heavychain and/or light chain from said B cell is expressed in a second cell.Said second cell is preferably used in order to produce a commerciallysuitable cell line.

The invention is further explained in the following examples. Theseexamples do not limit the scope of the invention, but merely serve toclarify the invention.

EXAMPLES Example 1

Methods

Human memory B cells are purified from peripheral blood or tonsil byfirst by positive selection for B cells with CD19 MACS® beads (MiltenyiBiotech). Memory B cells are then selected by surface staining and cellsorting for IgG, IgG⁺ B cells are then cultured with mouse fibroblast Lcells expressing CD40L in the presence of mouse or human IL-21 for 36 to48 hours. Cells are then transferred to Retronectin® (Takara, Shiga,Japan)—coated tissue culture plates where they are transduced with aretrovirus encoding human BCL6-IRES-GFP for 16 h at 37° C. Transducedcells are then cultured on CD40L-L, cells in the presence of human IL-2and human IL-4. After approximately 3-4 weeks the GFP⁺ cells (that is,BCL6⁺ cells) reach 100% of the culture after which BC16⁺ cells arecultured with IL-2 and IL-4 or with human or mouse IL-21. Using flowcytometry we monitor the expression of GFP, CD19, CD38, CD20, MHC classCD27 (BD Biosciences), and other markers using labeled antibodies. Wemonitor growth by cell counting, and Ig production is monitored byenzyme ELISA detection of Ig in the culture supernatant (Dako, Glostrup,Denmark), Gene expression is monitored by reverse transcriptasepolymerase chain reaction (RT-PCR, Invitrogen, Breda, Netherlands).

Results

Introduction of BCL6 into memory B cells results in a greatly extendedlifespan over normal B cells in culture (months vs. .about.3 weeks).These cells maintain CD19, surface Ig, NIFIC class II, and expressintermediate levels of CD38 and CD20, suggesting a memory cell phenotype(not shown). Culture of these cells on CD40L-L cells in the presence ofIL-21 results in a significant growth advantage (FIG. 1) and acquisitionof a plasmablast-like cell surface phenotype (CD38^(hi)D20⁺, FIG. 2),importantly, IL-21 cultured cells secrete 300% more IgG compared withcells cultured IL-2 and IL-4, Together these data show that IL-21culture promotes plasmablast development in an immortalized B cellpopulation, exhibiting enhanced growth and antibody production.

Example 2

A Non-Limiting Model of One Embodiment of the Present Invention isDepicted in FIG. 4.

In the human body, differentiation of plasma cells from memory B cellsinvolves downregulation BCL6 and upregulation of Blimp-1. In memorycells BCL6 is high and Blimp-1 expression is low. Signals that triggerdifferentiation cause an upregulation of Blimp-1, and this Blimp-1counteracts the expression of BCL6. This stage is short-lived and iscalled the plasmablast. With progressively increasing Blimp-1 levels,BCL6 expression is extinguished, resulting in a plasma cell.

In one embodiment of the invention BCL6 expression is “locked”, forinstance because of stable expression mediated by a retroviralexpression cassette integrated into the DNA of the B cells. Then, withBCL6 levels maintained, we “switch on” Blimp-1 expression, for instanceby use of a cytokine that activates STAT3, such as IL-21. Thiscombination, through modulation of key transcription, results in stablegrowth of cells that secrete antibody and have phenotype characteristicsof a plasmablast.

TABLE 1 Cell surface markers of memory B cells, plasmablasts and plasmacells Memory Plasmablast Plasma Cell CD38 + ++ ++ CD20 + + − CD27 + + −CD19 ++ + − CD138 − − + Proliferation low high none Ig secretion lowintermediate high BCL6 ++ + − Blimp-1 − + ++

Example 3

Materials and Methods

Maintenance and Isolation of Human B Cells.

Using standard procedures, CD19 positive human B cells were isolatedfrom bloodbank derived buffy coat (other sources can be fresh heparin orACD blood, or tonsil). In brief, total peripheral blood mononuclearcells (PBMC) were isolated using ficoll density separation (Amersham,Buckinghamshire, UK), CD19 labeled beads were used to positivelyselected B cells by MACS cell sorting technique (Miltenyi, Auburn,Calif., USA), Cells were subsequently stained with appropriatecombinations of monoclonal antibodies (mAbs) to CD19, CD27, IgG, IgM,CD3 (Becton Dickinson (BD), Franklin Lakes, N.J., USA) and phycoerythrin(PE) labeled Tetanus Toxoid (provided by A. Radbruch, Berlin, Germany)or any other labeled antigen. Cells were then sorted using the FACSAria(BD). Sorted cells were washed and cultured (1.5 to 2×10⁵ cells/ml) onirradiated CD40L-expressing L-cells (5×10⁴ cells/ml; provided by DR. J.Banchereau, Schering Plough France, Dardilly France), in Iscove'sModified D Minimal Essential Medium (IMDM) containing 8% fetal calfserum (FCS) and Penicillin/Streptomycin. Unless mentioned otherwise,these CD40L-expressing cells are always present in the cultures.

Transduction and Regulation of Mouse Constitutive Active STAT5b in BCells.

Purified B cells were primed to become transduced with the caSTAT5bgene. Two priming protocols were used: (1) purified B cells werecultured for 3 days with interleukin (IL) 2 (20 U/ml, Chiron,Emeryville, Calif., USA) followed by a 24 hour culture with IL-2 andIL-4 (10 ng/ml, R&D, Minneapolis, Minn., USA) or (2) purified B cellswere cultured for 36 hours with recombinant mouse IL-2 (50 ng/ml, R&D).Subsequently, cells were plated on recombinant human fibronectinfragments CH-296 (Hanenberg H., Nat., Med. 1996; RetroNectin, Takara,Japan) and human serum albumine treated plates (Corning Life Sciences,Corning, N.Y., USA) in the absence of L-cells, with the cytokines IL-2/4or IL-21. At last, cells were transduced with the caSTAT5b gene(described by Ariyoshi K., JBC, 2000 and obtained from T. Kitamura,IMSUT, Tokyo, Japan) fused to the estrogen receptor (ER, provided by H.Kurata, DNAX Institute, Palo Alto, Calif., USA). The activity of thecaSTAT5b-ER fusion product can be controlled by the hormonehydroxytarrioxifen (4HT, Sigma-Aldrich, St, Louis, Mo., USA). Thetransduction was performed using a retrovirus as described previously(Heemskerk M. H., JEM, 1997; Heemskerk M. H., Cell Immunol. 1999;Scheeren F. A., Nat Immunol, 2005). Transduction efficiency wasdetermined by antibody production of a truncated, signaling incompetentmutant of Nerve Growth Factor Receptor (.DELTA.NGFR, provided by C.Bonini, St. Raphael Hospital, Milan, Italy). Thus, outgrowth of B cellsthat contain the caSTAT5b gene depends on the presence of 4HT and thesecells can be detected by antibody staining for NGFR (Chromaprobe,Maryland Heights, Mo., USA).

Development of 100% caSTAT5b Positive B Cell Lines that SecreteAntibodies

We have developed a B cell line that produces monoclonal antibodies andis 100% caSTAT5b (=NGFR) positive. This was achieved by differentiatingB cells from a memory into an antibody producing phenotype, andtransducing with the caSTA5b-ER-IRES-NGFR construct. The action ofcaSTAT5b makes the differentiated B cells insensitive to cell death.Differentiation of B cells is induced in the first 2 to 3 weeks afterisolation (FIG. 5), using a cytokine mixture (IL-2, 4, 21 orcombinations of these cytokines and CD40L). The time point that caSTAT5bis activated by adding 4HT affects the overall phenotype of thecultures. This because eaSTAT5b blocks the cell to change its phenotypee.g. blocks further differentiation. Thus, the longer 4HT is withheldthe more B cells will differentiate into antibody producing cells orinto a type of cell that preferentially grows out under these cultureconditions (suggestions for cell types are: naive, follicular, memory,antibody producing, plasma blast, plasma cell, marginal zone,perisinusoidal or transitional B cellsmany of those B cell subsets haveonly been determined in mice). When 4HT is present in the culturemedium, caSTA5b-ER-IRES-NGFR positive B cells can survive for longperiods (Table 2).

TABLE 2 Overview of caSTAT5b-ER-IRES-NGFR transduced human B cellcultures. PBMC were obtained after Ficoll gradient isolation ofbloodbank derived buffy coats and subsequently sorted by CD19 MACS andCD27 or by FACSAria cell sorting. Purified B cells were then cultured inthe presence of L-cells with indicated cytokines before being transducedwith a retrovirus containing the caSTAT5b-ER-IRES-NGFR gene construct.Transduction Isolation Time on Culture Donor Date Subtype Protocol IL-21Time B12 28 Apr. 2005 CD19+TT+ IL-2 IL-4  4 wks 18 Oct. 2005 B15 17 May2005 CD19+TT+ IL-21 36 h 5 Dec. 2005 B16 31 May 2005 CD19+TT+ IL-21 20 d5 Dec. 2005 B18 22 Jun. 2005 CD19+CD27+ IL-2 IL-4 Time series 5 Dec.2005 and TT+ and IL-21 (36 h to 20 d) B19 22 Jun. 2005 CD19+CD27+ IL-2IL-4 Time series 5 Dec. 2005 and TT+ and IL-21 (36 h to 20 d) B20 6 Jul.2005 CD19+TT+ IL-21 36 h 5 Dec. 2005 B21 6 Jul. 2005 CD19+TT+ IL-21 36 h6 Sep. 2005 B22/B23/ 6 Sep. 2005 CD19+CD27+ IL-21  5 d 5 Dec. 2005 B24IgM− and TT+ B25/B26 20 Oct. 2005 CD19+CD27+ IL-21  7 d 5 Dec. 2005 andIgM− B27/B28 10 Nov. 2005 CD19+CD27+ IL-21  7 d 5 Dec. 2005 B29/B30 22Nov. 2005 CD19+CD27+ IL-21 42 h 5 Dec. 2005

Development of Single-Cell Derived, Clonal B Cell Cultures

Outgrowth of caSTA5b-ER-IRES-NGER positive B cells generally takes about4 weeks, after which clonal cultures can be obtained by performinglimiting dilution (LD) cultures or single cell sorting using flowcytometry (the FACSAria). These cultures consist of 2500 to 5000L-cells, normal concentrations of IL-2 and IL-4 and either 1, 5 or 10 Bcell/96-well when the LD is performed with 100% NGFR+ cells and 10, 100and 1000 cell/96-well when NGFR+ cells are sorted into 96 well using theFACSAria.

Restimulation of Antibody Production of caSTA5b-ER Positive B CellCultures

Poly-, oligo- or monoclonal caSTAT5b-ER-IRES-NGFR positive B cellcultures that were negative or low on antibody production were washedextensively before cultures were (1) deprived of 4HT, IL-2 and IL-4before being cultured with IL-21, then after 4-10 days of supernatantswere tested for IgM and IgG production or (2) deprived of 4HT for 10days meanwhile cultured with IL-2 and IL-4, and then at day 10 IL-2 andIL-4 are replaced by IL-21. Then at different time points supernatantsare tested for IgM and IgG production.

Results

B Cell Dyferentiation and Proliferation; the IL-2 and IL-4 vs. IL-21Protocol.

IL-21 treated B cell cultures showed enhanced proliferative responseswithin the first 2-3 weeks compared to IL-2 and IL-4 (FIG. 6a ).However, unlike the IL-2 and IL-4 cultures, continuous IL-21 stimulationresulted in decreased proliferation and cell death, even in the presenceof active STAT5b (FIG. 6b ). Suggesting that IL-21 eventually had to bereplaced by IL-2 and IL-4. To study this in more detail, time seriesexperiment were performed with CD19+CD27+ memory B cells, in which IL-21was replaced by IL-2 and IL-4 after 36 hours or 5, 10, 15 and 20 days.As shown in FIG. 7, most cultures could be maintained after IL-21withdrawal, even cultures that received IL-21 for 20 days.

Antibody Production by IL-21 Boosted Total Human Memory B Cell Cultures

Interestingly, in contrast to the IL-2 and IL-4 cultures, the IL-21boosted cultures were able to produce antibodies for a relatively longperiod (IgG and IgM as measured by ELISA, Dako, Glostrup, Denmark)(FIGS. 8a and 8b , respectively). Importantly, of the polyclonal memoryB cell cultures of donors B18 and B19, single-cell clones were obtainedby LD culture (table 3).

TABLE 3 Frequency of clones that were isolated from CD19+CD27+NGFR+sorted B cells. Cells were sorted in 96 wells at either 1000, 100, 10 or1 cell per well. Wells contained 5000 CD40L-expressing L cells, IL-2 andIL-4. ¼ to ½ of the medium was replaced twice a week with freshcytokines and 2500 L cells. B18 B19 Positive Total # Positive Total #Donor Well Wells Well Wells 1000 c/w 8 9 8 10 100 c/w 21 48 19 48 10 c/w6 48 2 48 1 c/w 1 96 6 96

The majority of the clonal cultures produced IgM while only someproduced IgG (FIG. 9). In addition, two clonal cultures produced bothIgM and IgG clone 7 and clone 8). Whether these clones are indeed clonalor that class switching occurred remains to be determined. In the latercase one BCR VDJ region should be found in the IgG or IgM gene fragmentsin this culture.

Antibody Production of IL-21 Boosted Tetanus Toxoid Specific B CellCultures.

Next, we tested whether we could isolate B cells producing TetanusToxoid (TT) specific antibodies. In brief, the following protocol wascarried out:

Part 1

-   (1) CD27+TT+ B cells were sorted (recovery was donor dependent and    ranged from 10000-1000 cells),-   (2) cultured with IL-21 for 36 h,-   (3) transduced with caSTA5b-ER-IRES-NGFR,-   (4) and cultured for variable times with IL-21 (36 h to 3 wks) after    which IL-21 was replaced by IL-2, IL-4 and 4HT.    Part 2-   (5) when cultures were 100% NGFR+ they were cloned by limiting    dilution (LD).

After 2 to 3 months of culture, 100% NGFR+α-TT-specific polyclonal Bcell cultures were obtained from at least 7 different donors (PART 1).All donors were tested positive in a α-TT-IgG specific antibody ELISA(r-biopharm, Darmstadt, Germany). As shown in FIG. 10a , α-TT IgG levelswere relatively low. Since immortalization of memory B cells resulted inhigh numbers of IgM producing cultures (FIG. 9), that indicates that themajority of the TT cultures are IgM positive too, Indeed, as shown inFIG. 10b , five out of seven donors were producing IgM, suggesting thatthe anti-TT antibodies are from IgM origin and thus not detected by ourα-TT IgG ELISA.

This let us to develop a α-TT IgM ELISA based on the r-biopharm TT IgGELISA. The only difference is in the final step, now a α-human IgM-HRPantibody instead of a anti-human IgG-HRP is added.

Next, from the polyclonal TT cultures, α-TT-specific B cell clones werederived by LD cultures (PART 2). These LD cultures were started with100% NGFR+ polyclonal .alpha.-TT-specific B cells from four donors(table 4). Clones from donor B16 mainly produced IgG, while B18 and B19produced IgM and B15 produced both IgG and IgM (not shown).Subsequently, supernatants of these clones were tested in the IgG TT orIgM TT ELISA (FIG. 11). Besides donor 15 all donors showed TT binding,although only 5 clones produced relatively high anti-TT antibody titers.

TABLE 4 Limiting dilution culture of 100% NGFR+ TT-specific B cells.Indicated is the total number of clones isolated and under whatconditions they were isolated, either 1, 5 or 10 cell/well. One 96 wellplate was used for each condition (1, 5 or 10 cell/well). Wellscontained 2500 CD40L-expressing L cells, IL-2 and IL-4. ¼ to ½ of themedium was replaced twice a week with fresh cytokines and 2500 L cells.Number of positive clones from 96 well Donor Total # From 1 c/w From 5c/w From 10 c.w B15 12 2 10 — B16 14 — 7 7 B18 10 10 — — B19 11 1 3 7

Restimulation of Antibody Production of IL-21 Boosted Tetanus ToxoidSpecific B Cell Cultures.

We were able to generate IgM and IgG producing poly- and monoclonal Bcell cultures using IL-21 as a stimulus. Nevertheless, antibodyproduction was not stable. To our surprise, however, these IL-21 treatedcultures could be restimulated to produce IgG and IgM antibodies (FIGS.12a and 12b , respectively). This was achieved by 4HT withdrawal andsimultaneously stimulation with IL-21. Using this protocol, totalantibody production increased 2- to 1000-fold for IgM, and 2- to 25-foldfor IgG. Several of the supernatants of restimulated monoclonal cultureswere now tested positive in the IgG and IgM Tetanus ELISA (FIG. 13).

Important to note is that caSTAT5b or caSTAT5b-ER B cell cultures thathad not been treated with IL-21 prior to caSTAT5b transduction andsubsequent expansion could not be restimulated to produce antibodiesunder any conditions (see patent application WO 03/052083; not shownhere),

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, Enhanced growth of BCL6⁺ cells cultured with IL-21. 100% pureBCL6⁺ memory B cells were cultured in the presence of IL-2 and IL-4(conventional culture conditions), or with IL-21 alone. The totalexpansion of live cells over 1 days of culture with IL-21 is shown, FIG.2, Plasmablast immortalization of BCL6-positive cells with IL-21, MemoryB cells were transduced with a retrovirus expressing BCL6-GFP andcultured with IL-2 and IL-4 (to prevent differentiation) or with IL-21for 14 days. The surface staining for CD38 and CD20 of GFP.sup.+ (thatis, BCL6.sup.+) cells is shown. IL-21 induces an 8-fold increase in theamount of B cells with a plasmablast phenotype.

FIG. 3. IL-21 upregulates BLIMP1 in B cells. 100% pure BCL6-.DELTA.NGFR⁺were cultured with IL-2 and IL-4 or IL-21 for 24 days, cDNA wasgenerated from total RNA and mRNA levels of BLIMP1 and HPRT (loadingcontrol) were determined by reverse transcriptase polymerase chainreaction,

FIG. 4, Non-limiting model of one embodiment according to the presentinvention.

FIG. 5 General overview of ideal culture scheme, see for more detailsthe material and methods section of Example 3.

FIG. 6a Growth dynamics of IL-2 and IL-4 vs. IL-21 stimulated. B cell.Peripheral blood (PB) memory B cells derived from two donors (B18 andB19) were stimulated either with IL-21 or IL-2 and IL-4. Cells weretransduced with caSTAT5b-ER-IRES-NGFR at day 2 for the IL-21 and at day5 for IL-2 and IL-4 treated cultures; 4HT was added at day 13.

FIG. 6b Of 4 donors Tetanus Toxoid specific B cells were sorted from PB(cell numbers ranged from 1000-10,000), Cells were cultured in 96 wellwith IL-21 and transduced with caSTAT5b-ER-IRES-NGFR on day 2. 4HT wasadded on day 4 and IL-21 was replaced with IL-2, IL-4 and 4HT after 7days (1314 and B15) or was replaced after 20 days (1316 and 1317), Cellswere counted by hand and dead cells were excluded.

FIG. 7 Percentage caSTAT5b-ER-IRES-NGFR transduced cells was determinedusing the LSR II (BD), Of two donors (B18 and B19) IL-2 and IL-4 vs.IL-21 time series experiment were performed, Of each donor ¼ of thecells were transduced using the IL-2 and IL-4 protocol, the remaining ¾was transduced using IL-21, Directly after the IL-21 transduction (36 h)one third of the IL-21 culture was switched to IL-2 and IL-4, This wasrepeated on day 5, 10 and 20 of the IL-21 culture,

FIG. 8a Total human IgG and IgM antibody production bycaSTAT5b-ER-IRES-NGFR transduced PB derived memory B cells. Identicalexperiment as described in FIG. 3, Mean IgG production of donor B18 andB19 is shown. B cells were transduced using the IL-2 and IL-4 vs. theIL-21 protocol. The IgG production indicated with the open symbolsrepresent all cultures that had been treated with IL-21, irrespectivewhen they were switched to IL-2 and IL-4.

FIG. 8b IgM production in samples as described above, note that the timescale is different,

FIG. 9 Antibody production of B cell clones derived from memory B cellsof donors 1318 and B19 transduced with caSTAT5b-ER-IRES-NGFR.Ten-day-old cultures that were derived from IL-21 stimulated B cells(stimulated for 36 h) were used for LI) culture. Twelve clones wereobtained; 5 from and 7 from 1319, IgG production is the mean of threetime points; IgM production is the mean of two time points.

FIG. 10a IgG Tetanus Toxoid ELISA on supernatant of polyclonal, 100%caSTA5b-ER-IRES-NGFR positive, Tetanus Toxoid sorted human B cells, Of 7donors rapidly proliferating clonal cultures were derived, Shown is theaverage antibody production of at least 3 different measurements perdonor, Each time the relative OD was determined (generally a relativeincrease of >2 to 3 times the background is assumed positive),

FIG. 10b To determine if TT IgG ELISA negative cultures could beproducing IgM, the same 7 donor samples were tested in a total IgMELISA.

FIG. 11 Anti-Tetanus Toxoid ELISA. The binding of IgG and IgM .alpha.-TTspecific antibodies by ELISA was determined, Supernatants of 100% NGFRpositive clonal B cell cultures derived from donors B15, 1316, B18 and1319 were tested. Two times the background was set as positive,

FIG. 12a Total IgG production after restimulation of clonal B cellcultures donor B16 which produces IgG. Production was measured insupernatant of cultures that were either cultured with IL-2, IL-4 and mthe presence or absence of 4HT or with IL-21 and in the presence orabsence of 4HT. Cultures containing IL-2 and IL-4 did not show anincrease in antibody secretion (not shown). Only cultures that respondedto the restimulation are shown (10 out of 14 IgG clones responded).

FIG. 12b Total IgM production after restimulation of clonal B cellcultures donor B19 which produces IgM, Production was measured insupernatant of cultures that were either cultured With IL-2, IL-4 and inthe presence or absence of 4HT or with IL-21 and in the presence orabsence of 4HT. Cultures containing IL-2 and IL-4 did not show anincrease in antibody secretion (not shown), Only cultures that respondedto the restimulation are shown 8 out of 9 IgM clones responded).

FIG. 13a Antibodies secreted by IL-21 restimulated and 4HT deprivedcultures, as described in the legend of FIG. 8 were tested for theirantigen specificity. The supernatants derived from restimulated donorB16 clonal IT cultures were tested in the .alpha.-TT IgG ELISA,

FIG. 13b As in FIG. 13a , here the supernatants derived from donor B19cultures were tested in the IgM ELISA. Shown is the relative increase inantibody binding compared to the negative control, samples B19-1087 and10E1 were cut off at 30 for visibility; values were 96 and 121,respectively. Shown is the relative increase in antibody bindingcompared to the negative control.

REFERENCES

-   Banchereau, J., de Paoli, P., Valle, A., Garcia, E., Rosset, F.,    (1991). Long term human B cell lines dependent on interleukin-4 and    antibody to CD40, Science 251, 70-2.-   Dadgostar, H., Zarnegar, B., Hoffmann, A., Qin, X. F., Truong, U.,    Rao, G., Baltimore, D., and Cheng, G. (2002). Cooperation of    multiple signaling pathways in CD40-regulated gene expression in B    lymphocytes, Proc. Natl. Acad. Sci USA 99, 1497-1502.-   Malisan, F., Briere, F., Bridon, J. M., Harindranath, N., Mills, F.    C., Max, E, E., Banchereau, J., Martinez-Valdez, H. (1996).    Interleukin-10 induces immunoglobulin G isotype switch recombination    in human CD40-activated naive B lymphocytes, J. Exp. Med. 183,    937-47.-   Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu,    Y., Gismondo, M. R., Murphy, B. R., Rappuoli, R., Lanzavecchiak. A.    (2004). An efficient method to make human monoclonal antibodies from    memory B cells: potent neutralization of SARS coronavirus. Nature    Medicine Volume 10, No. 8, 871-875.-   Ye, B. H., Cattoretti, G., Shen, Q., Zhang, S., Hawe, N., de Waard,    R., Leung, C., Nouri-Shirazi, M., Grazi, A., Chaganti, R. S., et al.    (1997). The BCL-6 proto-oncogene controls germinal-centre formation    and Th2-type inflammation, Nat Genet 16, 161-170.

The invention claimed is:
 1. A composition comprising: an isolated antibody producing cell that is stable for at least nine weeks, comprising an exogenous nucleic acid sequence encoding BCL6 or a functional part thereof; and IL-21 and/or IL-10.
 2. The composition of claim 1, comprising IL-21.
 3. The composition of claim 1, comprising an exogenous nucleic acid sequence encoding constitutively active BCL6.
 4. The composition of claim 1, wherein said cell is a human cell producing human antibodies.
 5. The composition of claim 1, wherein the antibody producing cell produces an antibody of interest.
 6. A composition comprising: an isolated antibody producing cell that is stable for at least nine weeks, comprising an exogenous nucleic acid sequence encoding BCL6 or a functional part thereof; and IL-21.
 7. An isolated antibody producing cell that is stable for at least nine weeks, comprising (i) an exogenous nucleic acid sequence encoding BCL6 or a functional part thereof; and (ii) IL-21. 